Method of quantifying tumour cells in a body fluid and a suitable test kit

ABSTRACT

A method for the quantification of tumor cells in a body fluid is disclosed and entails first carrying out a reaction with the sample to be investigated, in which the RNA component of telomerase is specifically amplified, and then the amount of amplified nucleic acid is determined quantitatively, as are test kits suitable therefor.

This application is the National Stage of International Application No.PCT/DE96/02183, filed Nov. 14, 1996. Benefit of priority to 35 U.S.C.§365(b) to German application no. 195 42 795.5, filed Nov. 16, 1995 isclaimed herein.

The invention relates to a method for the quantification of tumor cellsin a body fluid, in which firstly a reaction is carried out with thesample to be investigated, in which reaction the RNA component oftelomerase is specifically amplified, and subsequently the amount ofamplified nucleic acid is determined quantitatively, and to test kitssuitable therefor.

Virtually all solid malignant tumors have the potential to formmetastases. The metastasis process comprises the spread of malignantcells as micrometastases, usually via the blood or lymph to remoteorgans and the development of autonomous secondary tumors. The extent ofmetastasis determines the prognosis of an oncosis.

The requirements of tumor prevention or aftercare programs are todiagnose primary tumors or a recurrence or a metastasis early, evenbefore metastases become clinically manifest. This aim cannot yet besatisfactorily met with the available instrumental techniques; inparticular, there is still a diagnostic gray zone between circulatingtumor cells and incipient formation of metastases in organs. Earlydiagnosis of circulating malignant cells, for example in peripheralblood of a patient undergoing tumor aftercare would make it possible toapply immunomodulating therapy or polychemotherapy, which is possiblycurative, at an early date, that is to say even before organ metastasisbecomes manifest. Quantification of the metastases in peripheral bloodbefore and after the therapy represents an important control in suchcases.

GB 2 260 811 proposes, for example, a diagnostic method for detectingmalignant tumors which are associated with normal cells of a particularbody tissue, where the normal cells form at least one gene productspecific for this tissue. In this detection method, body fluid, forexample blood, in which the cells do not normally occur in a healthyperson, is taken from the patient, and the mRNA of the specific geneproduct is amplified and detected. An example mentioned is tyrosinasefor detecting melanoma cells in peripheral blood. However, thedisadvantage of this method is that it is linked to tissue-specific geneproducts, does not allow quantification of the melanoma cells and,moreover, gives false-positive results.

Kim et al. describes the results of an assay with which it was possibleto determine telomerase activities in tumor tissues [Kim et al. (1994).Science 266: 2011]. The telomerase activity was detected with asensitivity of about 1 immortal cell/104 normal cells in 98 of 100cancer cell cultures and 90 of 101 malignant tumors, and in germinaltissues, but not in 22 normal somatic cell-cultures.

Telomerase is a newly described ribonucleo-protein with reversetranscriptase activity [Shippen-Lentz et al. (1990), Science 247: 546]which uses an integral RNA sequence as template for independent DNAsynthesis [Greider et al. (1989). Nature 337: 331] by which newtelomeric DNA are synthesized at the ends of the chromosomes. Telomeresconsist of highly conserved (TTAGGG)n in tandem sequences with a lengthof about 5-15 kilobases (kb)/cell genome and have the task ofstabilizing the chromosomes on the nuclear membrane and protect thecoding genomic DNA from uncontrolled recombination and degradation[Mehle et al. (1994). Cancer Res 54: 236]. Whereas a dynamic equilibriumbetween shortening of the chromosome ends and de novo synthesis oftelomeric sequences by telomerase is postulated in lower eukaryotes,normal human somatic cells show low or undetectable telomerase activity.In addition, telomerase is not growth-regulated, in contrast to otherDNA enzymes, since none of the actively proliferating cell culturesshowed detectable telomerase activity. Only germ cells and almost alltumor cell lines [Ohyashiki et al. (1994). Cancer Genet Cytogenet 78:64;Rogalla et al. (1994). Cancer Genet Cytogenet 77: 19; Schwartz et al.(1995). Cancer 75: 1094] and tumor tissues (Lunge, [Hiyama et al.(1995). Oncogene 10: 937; Shirotani et al. (1994). Lung Cancer 11: 29],kidneys [Mehle et al. (1994). Cancer Res 54: 236], ovaries [Chadeneau etal. (1995). Cancer Res 55: 2533] and blood [Counter et al. (1995). Blood85: 2315]) show measurable telomerase activity and a constant telomerelength which is retained throughout an infinite number of celldivisions. Activation of telomerase with the stabilization, associatedtherewith, of the telomere length can therefore be regarded as acritical step in the direction of immortalization of somatic cells.

Feng et al. were able to clone the integral RNA sequence of humantelomerase (hTR), which is encoded on the distal segment (q) ofchromosome 3. The authors were able to demonstrate, by competitivepolymerase chain reaction (PCR), a significant increase in telomeraseexpression in tumor tissues and in germinal tissues by comparison withnormal somatic cells [Feng et al. (1995), Science 269: 1236]. Anantisense construct of the hTR sequence caused cell death (apoptosis) intransfected HeLa cells. These data demonstrate stringent repression oftelomerase in somatic tissues, as well as the fact that malignant growthdepends on the presence of immortal cells and on the activation oftelomerase.

The object of the present invention was therefore to develop a methodwith which it is possible to determine tumor cells quantitatively in abody fluid.

The invention therefore relates to a method for the quantification oftumor cells in a body fluid, in which firstly a reaction is carried outwith the sample to be investigated, in which reaction the RNA componentof telomerase is specifically amplified, and subsequently the amount ofamplified nucleic acid is determined quantitatively, and to test kitssuitable therefor. Body fluid means for the purpose of the presentinvention, for example, blood, urine or else stool, exudates ortransudates from body cavities, especially peripheral blood.

Peripheral blood is, for example, taken from the subject by puncturingan artery, vein or finger pad and is transferred into an RNA lysisbuffer which comprises, for example, urea or, preferably, guanidiniumisothiocyanate, in order to denature any RNases present and to releasethe nucleic acids from the cells [see, for example, Chomczynski et al.(1987) Anal. Biochem. 162, 156]. The nucleic acids can be isolated fromthe strongly saline medium of the RNA lysis buffer, for example, bymeans of silica particles to which all nucleic acids are able to bind[Boom et al. (1990) J. Clin. Microbiol., 29, 495]. The particles arethen washed several times with suitable buffer and the bound nucleicacids are eluted. It is subsequently advantageous to hydrolyze anygenomic DNA present in the sample using RNase-free DNase in a suitablebuffer, so that no false-positive results or excessive background noiseresult due to false amplification signals, because DNA is possibly stillpresent, in the later amplification of the RNA components of telomerase.This is generally followed by inactivation of the DNase, for example byphenol extraction and/or heat denaturation. It is possible andadvantageous, before or, preferably, after treatment of the sample withDNase, also to purify the RNA present in the sample further, for exampleby chromatographic methods such as ion exchange chromatography,preferably on silica gel.

To check whether possibly interfering genomic DNA is still present inthe sample, it is subsequently possible to carry out an amplificationreaction with the telomerase-specific oligonucleotide primers which aredescribed hereinafter, in which case the RNA present in the sample isnot transcribed to cDNA by a reverse transcription reaction beforehand.Only in the case where the sample is free of telomerase-specific DNAdoes no amplification take place, with the result that no amplified DNAcan be measured.

This is followed by transcription of the RNA present in the sample intocDNA, generally by means of the reverse transcription reaction, forexample with AMV reverse transcriptase. The method is generally knownand is described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, New York Cold Spring Harbor Laboratory, 1989. In apreferred embodiment of the reverse transcription, it is also possibleto use a thermostable RNA-dependent DNA polymerase as described in WO90/07641. Suitable oligonucleotide primers for the reverse transcriptaseare, for example and advantageously, the oligonucleotide primersdescribed below or random primers with a particular length.

The subsequent amplification can be carried out, for example, with a DNApolymerase, for example by the polymerase chain reaction (PCR) (see, forexample, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,965,188) or, preferably,with an RNA polymerase by, for example, isothermal nucleic acidsequence-based amplification (NASBA). Specific oligonucleotide primersderived from the nucleic acid to be amplified are required for theamplification in each case. It is possible in the present invention touse any sequence section of the RNA component of telomerase forsynthesizing the oligonucleotide primers. The oligonucleotide primersare preferably about 20 to about 30, preferably about 20 to about 25,nucleotides long. The amplification product is generally about 100 toabout 2000 bases, preferably about 200 to about 1500 bases, inparticular about 300 to about 350 bases, long. The followingoligonucleotide primers, which have been derived from the sequence shownin FIG. 1, are particularly preferred for the novel method:

5′ GACTCGGCTC ACACATGCAG TTCGC 3′ (TM1) (SEQ ID NO:1), and/or

5′ CTGGTCGAGA TCTACCTTGG GAGAAGC 3′ (TM2) (SEQ ID NO:2),

where TM1 and/or TM2 may, where appropriate, additionally comprise apromoter sequence for an RNA polymerase. The oligonucleotide primer TM1corresponds to the 5′ primer and TM2 corresponds to the 3′ primer. Theamplification product is 327 bp long. The primers may, for example, beprepared synthetically using the triester methods [Matteucci et al.,(1981), J. Am. Chem. Soc., 103, 3185-3191]. The DNA polymerase which canbe used is, for example, a non-thermostable DNA polymerase such as T4DNA polymerase, T7 DNA polymerase, E. coli polymerase I or the Klenowfragment of E. coli or, preferably, a thermostable DNA polymerase suchas Taq polymerase (see, for example, U.S. Pat. No. 4,889,818).

The general principle of the PCR consists of heat-denaturation of theDNA and restoration of the double strand in the presence of suitableoligonucleotide primers with opposite orientation of the single strandusing DNA polymerase in several repeated reaction cycles. The cycle isthen repeated until sufficient DNA has been formed for quantification byone of the methods described below. In general, about 20 to about 40cycles, preferably about 30 to about 35 cycles, suffice.

In the NASBA method (also called 3SR system) there is use of at leastone oligonucleotide primer, preferably TM2, which comprises a promoterfor the RNA polymerase, preferably for T7 RNA polymerase [see, forexample, Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA, 87,1874-1878 or Kievits et al. (1991), J. Virol. Methods, 35, 273-286].Firstly, as already described in detail above, the RNA is transcribedwith the aid of one of the oligonucleotide primers described above andof a reverse transcriptase, preferably AMV reverse transcriptase, intocDNA. The reaction product is an RNA:DNA double strand whose RNAcomponent is then degraded by an RNase, preferably RNase H, to give aDNA single strand. Using one of the oligonucleotide primers describedabove, the DNA single strand is made up to the DNA double strand using aDNA polymerase. AMV reverse transcriptase is once again a suitable andpreferred DNA polymerase because this transcriptase has not only anRNA-dependent DNA polymerase activity but also a DNA-dependent DNApolymerase activity. The reaction product is a DNA double strand whichcomprises the promoter for, for example, T7 RNA polymerase. The RNApolymerase then synthesizes an “antisense” RNA strand again, and thecycle can begin again. In general, about 20 to about 40 cycles,preferably about 30 to about 35 cycles, suffice to provide sufficientamplification product, preferably “antisense” RNA, for the subsequentquantification.

The amplification products can be quantified by, for example, stainingthem with ethidium bromide and detecting and quantifying them directly,for example, in an agarose or polyacrylamide gel. However, it isadvantageous for the amplification products to be labeled already duringthe amplification, because this achieves higher sensitivity. Examples ofsuitable labels are radioactive labels, biotin labels, fluorescent orelectrochemoluminescent (ECL) labels. The labels are, as a rule, carriedby the nucleotides as starting materials for amplification by DNA or RNApolymerase. The radiolabeled amplification products (for example PCR orNASBA products) can be detected by measuring the radioactivity, thebiotin-labeled amplification products can be detected via a dye whichcarries avidin or streptavidin, the fluorescent-labeled amplificationproducts can be detected with a fluorometer and theelectrochemoluminescent-labeled amplification products can be detectedwith an ECL detector. However, the most preferred detection method is invitro hybridization with a previously labeled oligonucleotide which iscomplementary to the amplification product. The oligonucleotidegenerally carries the labels described above, and in connection with theNASBA amplification method the hybridization probe carries anelectrochemoluminescent label, preferably atris[2,2-bipyridine]ruthenium[II] complex label [see, for example, vanGemen et al. (1994), J. Virol. Methods, 49, 157-168].

Accurate and sensitive quantification of the amplification products canadvantageously be achieved by coamplification of a defined amount of oneor more known nucleic acids (standard nucleic acids) [see, for example,van Gemen et al. (1993), J. Virol. Methods, 43, 177-188]. In this case,a different but exactly known amount of the coamplifying standardnucleic acid or nucleic acids is added, for example in the form of adilution series, to the unknown amounts of the sample to beinvestigated, and the amplification products of the sample and one ormore coamplifying standard nucleic acids are determined quantitativelyin independent mixtures. Comparison of the measured results reveals theamount of the RNA component of telomerase to be determined in thesample.

It is advantageous to amplify the coamplifying standard nucleic acid ornucleic acids with the same oligonucleotide primer as the sample to beinvestigated and the amplification products also have essentially thesame length. It is particularly preferred for the coamplifying nucleicacids to have the same sequence as the amplification product of thesample to be determined, with the exception of about 20 nucleic acidsbetween the oligonucleotide primer binding sites, which have anarbitrary but known sequence. It is possible thereby to quantify,independently of one another, the amplification product to be determinedin the sample from the coamplifying amplification product, for exampleby a hybridization as described in detail, for example, in Sambrook etal. (supra), using appropriately complementary labeled oligonucleotides.It is particularly advantageous if several, preferably three, differentcoamplifying nucleic acids are added in different concentrations to thesample, because this makes it possible to reduce the number ofindividual amplification reactions which would otherwise have to becarried out [see van Gemen et al. (1994) J. Virol. Methods 49, 157-168].It is also particularly preferred to add the coamplifying nucleic acidsto the RNA lysis buffer described above because it is possible therebyto exclude the effect of possible nucleic acid losses in the subsequentworkup of the sample.

Suitable and advantageous coamplifying standard nucleic acids in thepresent invention are RNA single stranded sequences which are preparedby in vitro transcription, for example with Sp6 or T7 RNA polymerase,from constructs which comprise the DNA or cDNA of the sample to beamplified and which are in each case provided with a randomized exchangeof a sequence of, for example, about 10 to about 30, preferably about20, nucleotides.

The constructs preferably consist of a transcription vector having abinding site for Sp6 or T7 RNA polymerase between a “multiple cloningsite” in which the DNA or cDNA of the sample to be amplified has beencloned. The cloned sequence can be opened by selective hydrolysis withrestriction endonucleases, preferably with two different restrictionendonucleases, and a fragment of a defined length can be cut out andreplaced by a fragment of equal length, for example using T4 ligase. Thecloned fragment may comprise replacement of a sequence of any length,for example about 10 to about 30, preferably about 20, nucleic acids andis preferably located between the oligonucleotide primer binding sites.This procedure can be repeated in order to insert other nucleic acidsequences at the same site. If no suitable cleavage sites can be found,for example because the vector is also cut, it is necessary to createartificial cleavage sites. This can take place, for example, byrecombinant PCR which is described in essence by Higuchi et al.[Higuchi, R. (1988). Nucleic Acid Res 16: 7351-7367; Higuchi, R. (1990).M. Innis A. et al. eds. San Diego, New York, Berkley, Boston, London,Sydney, Tokyo, Toronto, Academic Press, Inc. 177-183] and in theexperimental part of the present invention.

Preferably used for the purpose of the invention are in vitrotranscribed RNA single stranded sequences of constructs which

a) comprise the entire cDNA of the RNA component of human telomerase and

b) in which a randomized exchange of a sequence of about 20 nucleotideshas been introduced.

The constructs are derived from the constructs

pGEM-hTR shown in FIGS. 5a and 5 b (SEQ ID NO:17)

pGEM-hTR(Ka) shown in FIGS. 6a and 6 b (SEQ ID NO:18).

It is possible by in vitro transcription of the constructs with Sp6 RNApolymerase to prepare standard RNA nucleic acids which are individually975 base pairs long and have the sequence:

(hTRKa) shown in FIG. 7 (SEQ ID NO:19)

(hTRKb) shown in FIG. 8 (SEQ ID NO:20)

(htRKc) shown in FIG. 9 (SEQ ID NO:21).

The randomized sequence in which the standard nucleic acids differ fromthe wild-type RNA is in this example located in position 591-610 shownin FIG. 5a. It is 20 base pairs long.

Since the standard nucleic acids differ from one another and from thewild-type sequence in this example only by a randomized and knownsequence which is 20 base pairs long, the amplification products of thestandard nucleic acids and of the wild-type sequence can be detected bycomplementary binding of labeled oligonucleotides in four separatemixtures. Oligonucleotides which are particularly suitable for specificdetection of the amplified cDNA of the RNA component of telomerase (wt)and of the standard nucleic acids (hTRKa), (hTRKb) and (hTRKc) accordingto the present invention are the following sequences, which have beenderived from the sequences shown in FIGS. 7-9:

5′CGACTTTGGA GGTGCCTTCA 3′ O(wt) (SEQ ID NO:3) 5′AAGTCGGATC CACTTAGGTC3′ O(Ka) (SEQ ID NO:4) 5′CGCTCGATTT GGCGACGGGA 3′ O(Kb) (SEQ ID NO:5)5′GAGACTATAG CGATTGGACG 3′ O(Kc) (SEQ ID NO:6).

The corresponding reverse complementary sequences are used to detect theamplified “antisense” RNA.

After this, the individual amplified amounts of wild-type and standardnucleic acids are determined. The unknown initial amount of thewild-type sequence can be calculated by comparing with the differentamplified amounts of the standard nucleic acids when the initial amountis known (for example hTRKa: 10², hTRKb: 10⁴ and hTRKc: 10⁶ molecules).It is then possible to conclude from this the number of metastases forthe removed body fluid.

As internal positive control of the method and of the sample to beinvestigated it is possible additionally to amplify and detect a nucleicacid which generally always occurs in a body fluid. Examples of suitablenucleic acids are the mRNA coding for β-globin or forglyceraldehyde-phosphate dehydrogenase (GAPDH) (see, for example, GB 2260 811) which always occur in the cells of the body fluid. Suitableoligonucleotide primers for human β-globin mRNA are, for example,primers with the sequences:

5′ACCCAGAGGT TCTTTGAGTC 3′ and (SEQ ID NO:7) 5′TCTGATAGGC AGCCTGCACT 3′(SEQ ID NO:8),

where the oligonucleotide primers may, where appropriate, additionallycomprise a promoter sequence for an RNA polymerase.

To prevent or reduce false-positive results or so-called backgroundnoise which is caused by telomerase activities which are possiblypresent in nontumor cells, it is advantageous to purify the body fluidwhich has been taken before the novel investigation. The intention is,in particular, to deplete stem cells and/or activated immune cells, orconcentrate tumor cells, in the sample to be investigated. Since, as arule, the individual cells have specific surface markers, removal orconcentration of the cells by immunoabsorption is particularlyadvantageous. Examples of suitable methods are magnetic (MACS) orfluorescence-activated (FACS) cell sortings [see, for example,Göttlinger & Radbruch (1993) mta, 8, 530-536, No. 5]. Thus, for example,hemopoietic stem cells can be removed from the blood sample by means ofMACS via their CD34 surface marker [Kato & Radbruch (1993) Cytometry,14, 384-392]. B cells can be removed, for example, via their CD10, CD19and/or CD20 surface markers, and T cells via CD45RA and/or CD7. Tumorcells can be concentrated via their specific tumor markers, for exampleCEA. Besides MACS or FACS, also particularly suitable for depletion orconcentration of the relevant cells are antibodies against the specificsurface markers, which are bound in particular to commerciallyobtainable magnetic beads (for example Dynabeads M450, Dynal Corp.).

It is also particularly advantageous, alone or in conjunction with thepurification methods described above, to compare the amount of RNAcomponent of telomerase from venous blood with the amount of RNAcomponent of telomerase from arterial blood, since it is possible todetect, for the purpose of tumor cell determination, only about 20% ofall cells in venous blood samples, compared with 100% of the cells inarterial blood samples [Koop, S. et al. (1995) Cancer Res. 55,2520-2523]. It is likewise suitable to compare blood from the finger padwith venous or arterial blood.

Quantitative determination of the RNA component of telomerase in thesample makes it possible to determine whether tumor cells, especiallymetastases, in particular micrometastases, of malignant tumors arepresent in the body fluid, and in what quantity. This is of great use inparticular for early clinical diagnosis of the formation of metastasesfrom malignant tumors and for monitoring tumor therapy. Tumor cellswhich can be detected with the present invention are, in particular,tumor cells from metastases, preferably micrometastases, from malignanttumors, especially cells from metastasizing tumors and/or neoplasmswhich are derived, for example, from a T-cell lymphoblastoma, T-cellleukemia cells, chronic myeloid leukemia cells, acute lymphatic leukemiacells, chronic lymphatic leukemia cells, teratocarcinoma, melanoma,carcinoma of the lung, large bowel cancer, breast cancer, hepatocellularcarcinoma, kidney tumor, adrenal tumor, prostate carcinoma,neuroblastoma, brain tumor, small-cell carcinoma of the lung,rhabdomyosarcoma, leiomyosarcoma and/or lymphoma.

The present invention further relates to the oligonucleotide primerswith the sequence

5′ GACTCGGCTC ACACATGCAG TTCGC 3′ (TM1) (SEQ ID NO:1), 5′ CTGGTCGAGATCTACCTTGG GAGAAGC 3′ (TM2) (SEQ ID NO:2), 5′ ATAAGAATGC GGCCGCGGGTTGCGGAGGGT GGGCCTGGGA GGG 3′ (TMK1) (SEQ ID NO:9), 5′ CCCAAGCTTGTGGGGGTTAT ATCCTA 3′ (TMK2) (SEQ ID NO:10), 5′ CGCGGATCCA CTTAGGTCATCGATCTGCCA ATTTGCAGCA CACT 3′ (TMK3) (SEQ ID NO:11) and/or 5′ CGCGGATCCGACTTGGTACC ATGAATGGGC AGTGAGCCGG 3′ (TMK4) (SEQ ID NO:12),

where TM1 and/or TM2 may, where appropriate, additionally comprise apromoter sequence for an RNA polymerase;

and

an oligonucleotide with the sequence

5′ CCATCGATTC CCGTCGCCAA ATCGAGCGGG TACCCC 3′ (Kb) (SEQ ID NO:13) 3′GGTAGCTAAG GGCAGCGGTT TAGCTCGCCC ATGGGG 5′, or 5′ CCATCGATCG TCCAATCGCTATACTCTCGG TACCCC 3′ (Kc) (SEQ ID NO:14) 3′ GGTAGCTAGC AGGTTAGCGATATGAGAGCC ATGGGG 5′;

and

a nucleic acid construct pGEM-hTR as shown in FIGS. 5a and 5 b or anucleic acid construct pGEM-hTR(Ka) as shown in FIGS. 6a and 6 b;

and the standard RNA nucleic acid for coamplification of the sequence:

(hTRKa) as shown in FIG. 7 (SEQ ID NO:19)

(hTRKb) as shown in FIG. 8 (SEQ ID NO:20)

(hTRKc) as shown in FIG. 9 (SEQ ID NO:21), and the cDNAs thereof, andthe oligonucleotides for detecting the amplified cDNA of the wild-typenucleic acid and of the cDNA of the hTRKa, hTRKb and hTRKc standardnucleic acids with the sequence:

5′ CGACTTTGGA GGTGCCTTCA 3′ O(wt) (SEQ ID NO:3) 5′ AAGTCGGATC CACTTAGGTC3′ O(Ka) (SEQ ID NO:4) 5′ CGCTCGATTT GGCGACGGGA 3′ O(Kb) (SEQ ID NO:5)5′ GAGAGTATAG CGATTGGACG 3′ O(Kc) (SEQ ID NO:6)

and the corresponding reverse complementary sequences of theoligonucleotides for detecting the amplified “antisense” RNA.

The invention additionally relates to a kit for quantifying tumor cellsin a body fluid, for example blood, urine or else stool, exudates ortransudates from body cavities, especially peripheral blood, comprising

(a) nucleic acid or nucleic acids for the coamplification, and

(b) oligonucleotide primer pair for specific amplification oftelomerase-encoding nucleic acid and of the nucleic acid or nucleicacids specified in (a), where the standard RNA nucleic acid for thecoamplification mentioned in (A) has or have the following sequence:

(hTRKa) as shown in FIG. 7 (SEQ ID NO:19)

(hTRKb) as shown in FIG. 8 (SEQ ID NO:20)

(hTRKc) as shown in FIG. 9 (SEQ ID NO:21),

and/or the oligonucleotide primer pair preferably the followingsequences:

5′ GACTCGGCTC ACACATGCAG TTCGC 3′ (TM1) and/or (SEQ ID NO:1) 5′CTGGTCGAGA TCTACCTTGG GAGAAGC 3′ (TM2) (SEQ ID NO:2),

where TM1 and/or TM2 may, where appropriate, additionally comprise apromoter sequence for an RNA polymerase.

The novel kit may also comprise in addition, as described in detailabove, a labeled oligonucleotide as hybridization probe for specificdetection of the amplified cDNA of the wild-type sequence and/or severallabeled oligonucleotides as hybridization probe for specific detectionof the amplified cDNA of the standard nucleic acid or nucleic acids. Inaddition, a novel kit for PCR amplification may additionally comprisethe enzymes described in detail above, where appropriate labelednucleotides and/or suitable buffers, such as, for example, a reversetranscriptase, preferably an AMV reverse transcriptase, a DNApolymerase, preferably a Taq polymerase and/or a DNase and, whereappropriate, means suitable for depletion of stem cells and/or activatedimmune cells and/or for concentration of tumor cells, as described indetail above.

Another novel kit for NASBA may likewise comprise, besides the standardnucleic acids described in detail above, a labeled oligonucleotide ashybridization probe for specific detection of the amplified “antisense”RNA of the wild-type sequence and/or several labeled oligonucleotides ashybridization probe for specific detection of the amplified “antisense”RNA of the standard nucleic acid or nucleic acids. It may additionallylikewise comprise the enzymes described in detail above, whereappropriate labeled nucleotides and/or suitable buffers, such as, forexample, a reverse transcriptase, preferably an AMV reversetranscriptase, an RNA polymerase, preferably a T7 RNA polymerase, anRNase H and/or a DNase, and, where appropriate, means suitable fordepletion of stem cells and/or activated immune cells and/or forconcentration of tumor cells, as described in detail above.

The following examples and figures are intended to describe the presentinvention in detail without, however, restricting it thereto.

DESCRIPTION OF THE FIGURES

FIG. 1 (SEQ ID NO:16) shows the RNA component of human telomerase andthe position of the designed oligonucleotide primers: 5′ primer TM1(position 428-452) and 3′ primer TM2 (position 728-754) with anamplification product of 327 base pairs (bp) or 5′ primer TMK1 ([16bp]+1-27) and 3′ primer TMK2 (position 940-962+[3 bp]) with anamplification product of 981 bp. Hydrolysis with the restrictionendonucleases NotI and HindIII give the 962 bp fragment hTR.

FIG. 2 shows a PCR amplification on the cDNA of tumor cell lines.

Bands 1: MT4, T-cell lymphoblastoma cell line, bands 2: C8166, T-cellleukemia cell line, bands 3: K562, chronic myeloid leukemia (CML) cellline, bands 4: Molt4, acute lymphatic leukemia (ALL) cell line and bands5: teratocarcinoma cell line; M: 100 bp marker. hTR: RT-PCR with the TM1and TM2 primers; hTR/φRT: control PCR without reverse transcription (RT)reaction, GAPDH: control RT-PCR with primers forglyceraldehyde-phosphate dehydrogenase (GAPDH).

FIG. 3 shows a PCR amplification with the TM1 and TM2 primers on thecDNA from tumor tissues and healthy reference tissues. M: 100 bp marker;band 1: kidney carcinoma, band 2: healthy kidney tissue; band 3: thyroidcarcinoma, band 4: healthy thyroid tissue, band 5: carcinoma of breast,band 6: healthy breast tissue; band 7: colon carcinoma, band 8: healthylarge bowel tissue; band 9: H₂O control reaction.

FIG. 4 shows a PCR amplification with the TM1 and TM2 primers on thecDNA from peripheral blood of normal subjects and leukemia patients. M:100 bp marker; bands 1-3: healthy blood donors; bands 4-8: patients withacute myeloid leukemia (AML); band 9: H₂O control reaction.

FIGS. 5a and 5 b (SEQ ID NO:17) show the construct pGEM-hTR (4118 bp)with the transcription vector pGEM-13Zf(+) and the fragmenthTR(NotI/HindIII) shown in FIG. 1, which comprises the cDNA of the RNAcomponent of human telomerase (bases 1-956: position 12-975). Theposition of the NotI addition (position: 12-17) by the oligonucleotideprimer TMK1 is shown by dotted line.

FIGS. 6a and 6 b (SEQ ID NO:18) show the construct pGEM-hTR(Ka) (4118bp) with the ClaI-BamHI-KpnI cassette (position: 585-616) and thepositions of the designed oligonucleotide primers (5′ primer TMK1:position [16 bp]+1-27, 3′ primer TMK3: position 565-605+[24 bp]) with anamplification product of 606 bp, (5′ primer TMK4: position 60-636+[20bp], 3′ primer TMK2: position 940-962+[3 bp]) with an amplificationproduct of 387 bp. Hydrolysis with the restriction endonucleases NotIand BamHI or BamHI and HindIII gives a product of 588 or 375 bprespectively. Ligation of the fragments in pGEM-13Zf(+) gives a productof 963 bp.

FIG. 7 (SEQ ID NO:19) shows the standard RNA hTRKa (975 bp) after invitro transcription with Sp6 RNA polymerase on the construct pGEM-hTRKa,which has been linearized with HindIII, and the position of therandomized sequence of 20 bp (position 591-610).

FIG. 8 (SEQ ID NO:20) shows the standard RNA hTRKb (975 bp) after invitro transcription with Sp6 RNA polymerase on the construct pGEM-hTRKb,which has been linearized with HindIII, and the position of therandomized sequence of 20 bp (position 591-610).

FIG. 9 (SEQ ID NO:21) shows the standard RNA hTRKc (975 bp) after invitro transcription with Sp6 RNA polymerase on the construct pGEM-hTRKc,which has been linearized with HindIII, and the position of therandomized sequence of 20 bp (position 591-610).

EXAMPLES

Unless noted otherwise, the following examples were carried out bystandard methods as described, for example, by Sambrook, J. et al.(1989) supra, or in accordance with the instructions of themanufacturers of the kits and enzymes used.

1. Cultivation and isolation of peripheral blood, tissue and cells

Tumor cell lines such as MT4 (T-cell lymphoblastoma), C8166 (T-cellleukemia), K562 (chronic myeloid leukemia (CML)), Molt4 (acute lymphaticleukemia (ALL)) and teratocarcinoma were cultured as recommended by theATCC (American Tissue Culture Collection). Venous blood donated byleukemia patients (acute myeloid leukemia) and healthy control subjectswas taken by puncture of a forearm vein in EDTA-monovets^(®) (Saarsted).Human tumor tissue and healthy reference tissue were shock-frozenimmediately after removal in liquid nitrogen and stored at −70° C.

2. Isolation of cellular RNA

Total cellular RNA was isolated by standard methods [Chomczynski et al.(1987) Anal Biochem 162, 156; Sambrook, J. et al. (1989). Cold SpringHarbor, N.Y., USA, Cold Spring Harbor Laboratory Press]. Peripheralblood was transferred immediately after removal into RNA lysis buffer (4M guanidinium isothiocyanate; 0.1 M Tris-HCl, pH 7.5; 1%mercaptoethanol). Tissues and cells were homogenized in RNA lysis bufferusing an Ultra-Turrax T25 dispersing apparatus (Laborreaktor-Systeme,IKA). The mixtures were either immediately processed further or storedat −70° C.

3. Reverse transcription and polymerase chain reaction (RT-PCR)

The RT-PCR was carried out with the GeneAmp^(®) RNA-PCR kit (PerkinElmer) as specified by the manufacturer. Aliquots of the isolated totalRNA from peripheral blood, cell lines and tissues were in each casepreviously hydrolyzed with 20 U of DNase (Boehringer, Mannheim) in 10 μlmixtures (in 50 mM KCl; 10 mM Tris-HCl, pH 8.3 and 2.5 mM MgCl₂) at 37°C. for 60 minutes and then the DNase was inactivated at 95° C. for 30minutes. For complete purification of the RNA from proteins and DNAfragments, the DNase hydrolysates were in each case purified again on asilica gel matrix (RNeasy^(®), Qiagen) and measured by photometry.

The two oligonucleotide primers:

TM1 5′ GACTCGGCTC ACACATGCAG TTCGC 3′ (SEQ ID NO:1)

TM2 5′ CTGGTCGAGA TCTACCTTGG GAGAAGC 3′ (SEQ ID NO:2) were designed inaccordance with the sequence, published by Feng et al., of the RNAcomponent of human telomerase (Feng, J. et al. (1995). Science 269:1236-41) (FIG. 1) and synthesized using an Applied Biosystems 380Asynthesizer. The specificity of the TM1 and TM2 primers was checked bycomputer-assisted analysis of homology on the nucleic acid sequences inthe GenBank, EMBL, DDBJ and PDB databanks using BLASTN 1.4.9 MP[Altschul, S. F. et al. (1990). J Mol Biol 215: 403-410].

For consistency of the amplified amounts, the same amounts of RNA wereemployed for the RT reaction in each experiment. In order to precludecontamination of the RNA preparations with genomic DNA, eachRNA-containing sample hydrolyzed with DNase was first subjected to thePCR described below and checked for amplification. The RNA-containingsample in which no amplification product was detectable was employed forthe subsequent cDNA synthesis and PCR steps. Oligonucleotide primers forGAPDH were employed as internal standard control.

The PCR was carried out on 5 μl of the cDNA reaction or on a 1:50dilution of the isolated plasmid DNA in accordance with the followingprogram: (95° C.: 2 minutes, preheating); (94° C.: 30 seconds, 60° C.:30 seconds, 72° C.: 30 seconds, 35 cycles); (72° C.: 7 minutes, finalextension). The amplification products were fractionated by gelelectrophoresis on 1.5% TAE agarose gel, stained with ethidium bromideand visualized and recorded by photography under UV light (see FIGS.2-4).

4. Preparation of the standard RNA nucleic acids hTRKa, hTRKb and hTRKc

The enzymes used, such as Sp6 RNA polymerase, T4 ligase and restrictionendonucleases, inter alia from Boehringer Mannheim, Biolabs and Promega,were used as recommended by the manufacturers. The PCR amplificationproducts intended for cloning were fractionated by gel electrophoresison 1.5% TAE agarose and eluted (Qiagen). The restriction hydrolysateswere purified by phenol/chloroform extraction and precipitated in saltand ethanol or by DNA purification (Qiagen). The constructs were clonedby ligating the fragments into the corresponding cleavage sites in thecloning and transcription vector pGEM-13Zf(+) using T4 ligase. Thisvector permits in vitro transcription of cloned fragments by use of Sp6or T7 RNA polymerases as selected. Competent bacteria (XL-1Blue,Stratagene) were transformed by electroporation (BioRad). Plasmid DNAwas purified using plasmid purification kits (Qiagen). Positive cloneswere validated using vector- or sequence-specific oligonucleotideprimers with the PCR. Sequence validation was carried out for theconstructs by semiautomatic sequence analysis.

The construct pGEM-hTR (FIGS. 5a and 5 b) was created as initialconstruct for the constructs pGEM-hTR(Ka) (FIGS. 6a and 6 b),pGEM-hTR(Kb) and pGEM-hTR(Kc). pGEM-hTR(Ka) differs from pGEM-hTR by arandomized exchange of sequence in position 585-616. The constructspGEM-hTRKb and pGEM-hTRKc differ from pGEM-hTR by a randomized sequenceexchange in position 587-615. The constructs were used for in vitrotranscription with Sp6 RNA polymerase of the standard RNA: hTRKa (FIG.7), hTRKb (FIG. 8) and hTRKc (FIG. 9). To form the construct pGEM-hTR,the cDNA of the RNA component of human telomerase hTR (FIG. 1) wascloned into the NotI and HindIII cleavage sites of pGEM-13Zf(+). Thiswas achieved by carrying out an RT-PCR with the followingoligonucleotide primers, which were derived from the sequence hTR (FIG.1),

5′ ATAAGAATGC GGCCGCGGGT TGCGGAGGGT GGGCCTGGGA GGG 3′ (TMK1) (SEQ IDNO:9) 5′ CCCAAGCTTG TGGGGGTTAT ATCCTA 3′ (TMK2) (SEQ ID NO:10)

on the previously isolated RNA from tumor cells or lines under theconditions described above. The oligonucleotide primer TMK1 (position1-27, FIG. 1) contains an additional 5′ extension of 16 bp and a NotIcleavage site, and the oligonucleotide primer TMK2 (position 940-962,FIG. 1) contains an additional 3 bp extension and a HindIII cleavagesite. The TMK1 and TMK2 primer pair amplifies a 979 bp fragment. After arestriction hydrolysis with NotI and HindIII, the 963 bp fragmenthTR(NotI-HindIII) (position 1-957, FIG. 5) was cloned into thecorresponding cleavage sites (position 12 and 38) of pGEM-13Zf(+), andthe 4118 bp construct pGEM-hTR was created. pGEM-hTR(Ka) was constructedby replacing a 32 bp sequence in the construct pGEM-hTR (position585-616) by a 32 bp ClaI-BamHI-KpnI cassette:

5′ ATCGATGACC TAAGTGGATC CGACTTGGTA CC 3′ (SEQ ID NO:15) 3′ TAGCTACTGGATTCACCTAG GCTGAACCAT GG 5′.

This replacement was carried out by recombinant PCR and is amodification of the method described by Higuchi et al. [Higuchi, R.(1988). Nucleic Acid Res 16: 7351-7367; Higuchi, R. (1990). M. Innis A.et al. eds. San Diego, New York, Berkley, Boston, London, Sydney, Tokyo,Toronto, Academic Press, Inc. 177-183]. In a first step, two independentPCRs were carried out on pGEM-hTR:

The 1^(st) PCR took place with the following oligonucleotide primers,which were derived from the sequence hTR (FIG. 1):

5′ ATAAGAATGC GGCCGCGGGT TGCGGAGGGT GGGCCTGGGA GGG 3′ (TMK1) (SEQ IDNO:9 5′ CGCGGATCCA CTTAGGTCAT CGATCTGCCA ATTTGCAGCA CACT 3′ (TMK3) (SEQID NO:11)

The oligonucleotide primer TMK3 (position 565-605, FIG. 6) contains anadditional 5′ extension of 24 bp with a BamHI and ClaI cleavage site andencodes 21 bp of the ClaI-BamHI-KpnI cassette. The amplification productfrom the 1^(st) PCR gives the 5′ fragment of 606 bp and was digestedwith NotI and BamHI to give a 588 bp 5′ fragment.

The 2^(nd) PCR took place with the following oligonucleotide primers,which were derived from the sequence hTR (FIG. 1):

5′ CGCGGATCCG ACTTGGTACC ATGAATGGGC AGTGAGCCGG 3′ (TMK4) (SEQ ID NO:12)5′ CCCAAGCTTG TGGGGGTTAT ATCCTA 3′ (TMK2) (SEQ ID NO:10).

The oligonucleotide primer TMK4 (position 599-618, FIG. 6) contains anadditional 5′ extension of 20 bp with a BamHI and KpnI cleavage site andencodes 17 bp of the ClaI-BamHI-KpnI cassette. The amplification productfrom the 2^(nd) PCR gives the 3′ fragment of 387 bp and was hydrolyzedwith BamHI and HindIII to give a 375 bp 3′ fragment. T4 ligase was usedto connect the BamHI cleavage sites of the 5′ and 3′ fragments togetherto give the 963 bp NotI-HindIII fragment, which was cloned into thecorresponding cleavage sites (position 12 and 38) of pGEM-13Zf(+) tocreate the 4118 bp construct pGEM-hTR(Ka) (FIG. 6). pGEM-hTR(Kb) andpGEM-hTR(Kc) were constructed by replacing a 29 bp sequence in theconstruct pGEM-hTR(Ka) (position 587-615) by a randomized sequence of 29bp. A selective restriction digestion with ClaI and KpnI on theconstruct pGEM-hTR(Ka) and the following oligonucleotides Kb and Kc

      ClaI                          KpnI (SEQ ID NO:13) 5′ CCATCGATTCCCGTCGCCAA ATCGAGCGGG TACCCC 3′ Kb 3′ GGTAGCTAAG GGCAGCGGTT TAGCTCGCCCATGGGG 5′       ClaI                          KpnI (SEQ ID NO:14) 5′CCATCGATCG TCCAATCGCT ATACTCTCGG TACCCC 3′ Kc 3′ GGTAGCTAGC AGGTTAGCGATATGAGAGCC ATGGGG 5′

was followed by cloning, in two separate T4 ligase reactions, theClaI-KpnI fragment of the oligonucleotides Kb and Kc into thecorresponding cleavage sites of pGEM-hTR(Ka) to create the 4118 bpconstructs pGEM-hTR(Kb) and pGEM-hTR(Kc).

RNA was produced in vitro in a length of 975 bp from pGEM-hTR(Ka)(hTRKa, FIG. 7), pGEM-hTR(Kb) (hTRKb, FIG. 8) and pGEM-hTR(Kc) (hTRKc,FIG. 9) with Sp6 RNA polymerase. The further processing of the RNA, suchas DNase digestion, purification and calibration, took place by standardmethods.

5. Results

The investigations on tumor cell lines revealed that the RNA componentof human telomerase was detectable in different amounts in all the tumorcell lines with the same amount amplified in the GAPDH control reaction(FIG. 2). It was possible to rule out contamination with genomic DNA ineach case by a control reaction without addition of reversetranscriptase.

The comparative investigations on tumor tissue and healthy tissuerevealed that the RNA component of human telomerase could be detectedunambiguously in tumor tissues but not in healthy reference tissues(FIG. 3). The variation in intensity of the amplification products canbe explained by the individual quality of the RNA obtained from thetumor tissues.

The investigations with venous blood revealed that different levels oftelomerase activities were detectable in blood from healthy subjects andfrom leukemia patients, with distinctly lower telomerase activitiesbeing found in the control subjects by comparison with the cancerpatients (FIG. 4).

The in vitro transcription using Sp6 RNA polymerase on the constructspGEM-hTR(Ka), pGEM-hTR(Kb) and pGEM-hTR(Kc) revealed in each case therequired RNA transcript hTRKa, hTRKb and hTRKc with a length of 975bases.

21 25 base pairs nucleic acid single linear Genomic DNA 1 GACTCGGCTCACACATGCAG TTCGC 25 27 base pairs nucleic acid single linear Genomic DNA2 CTGGTCGAGA TCTACCTTGG GAGAAGC 27 20 base pairs nucleic acid singlelinear Genomic DNA 3 CGACTTTGGA GGTGCCTTCA 20 20 base pairs nucleic acidsingle linear Genomic DNA 4 AAGTCGGATC CACTTAGGTC 20 20 base pairsnucleic acid single linear Genomic DNA 5 CGCTCGATTT GGCGACGGGA 20 20base pairs nucleic acid single linear Genomic DNA 6 GAGAGTATAGCGATTGGACG 20 20 base pairs nucleic acid single linear Genomic DNA 7ACCCAGAGGT TCTTTGAGTC 20 20 base pairs nucleic acid single linearGenomic DNA 8 TCTGATAGGC AGCCTGCACT 20 43 base pairs nucleic acid singlelinear Genomic DNA 9 ATAAGAATGC GGCCGCGGGT TGCGGAGGGT GGGCCTGGGA GGG 4326 base pairs nucleic acid single linear Genomic DNA 10 CCCAAGCTTGTGGGGGTTAT ATCCTA 26 44 base pairs nucleic acid single linear GenomicDNA 11 CGCGGATCCA CTTAGGTCAT CGATCTGCCA ATTTGCAGCA CACT 44 40 base pairsnucleic acid single linear Genomic DNA 12 CGCGGATCCG ACTTGGTACCATGAATGGGC AGTGAGCCGG 40 36 base pairs nucleic acid double linearGenomic DNA 13 CCATCGATTC CCGTCGCCAA ATCGAGCGGG TACCCC 36 36 base pairsnucleic acid double linear Genomic DNA 14 CCATCGATCG TCCAATCGCTATACTCTCGG TACCCC 36 32 base pairs nucleic acid double linear GenomicDNA 15 ATCGATGACC TAAGTGGATC CGACTTGGTA CC 32 962 base pairs nucleicacid single linear Genomic DNA 16 GGGTTGCGGA GGGTGGGCCT GGGAGGGGTGGTGGCCATTT TTTGTCTAAC CCTAACTGAG 60 AAGGGCGTAG GCGCCGTGCT TTTGCTCCCCGCGCGCTGTT TTTCTCGCTG ACTTTCAGCG 120 GGCGGAAAAG CCTCGGCCTG CCGCCTTCCACCGTTCATTC TAGAGCAAAC AAAAAATGTC 180 AGCTGCTGGC CCGTTCGCCT CCCGGGGACCTGCGGCGGGT CGCCTGCCCA GCCCCCGAAC 240 CCCGCCTGGA GCCGCGGTCG GCCCGGGGCTTCTCCGGAGG CACCCACTGC CACCGCGAAG 300 AGTTGGGCTC TGTCAGCCGC GGGTCTCTCGGGGGCGAGGG CGAGGTTCAC CGTTTCAGGC 360 CGCAGGAAGA GGAACGGAGC GAGTCCCGCCGCGGCGCGAT TCCCTGAGCT GTGGGACGTG 420 CACCCAGGAC TCGGCTCACA CATGCAGTTCGCTTTCCTGT TGGTGGGGGG AACGCCGATC 480 GTGCGCATCC GTCACCCCTC GCCGGCAGTGGGGGCTTGTG AACCCCCAAA CCTGACTGAC 540 TGGGCCAGTG TGCTGCAAAT TGGCAGGAGACGTGAAGGCA CCTCCAAAGT CGGCCAAAAT 600 GAATGGGCAG TGAGCCGGGG TTGCCTGGAGCCGTTCCTGC GTGGGTTCTC CCGTCTTCCG 660 CTTTTTGTTG CCTTTTATGG TTGTATTACAACTTAGTTCC TGCTCTGCAG ATTTTGTTGA 720 GGTTTTTGCT TCTCCCAAGG TAGATCTCGACCAGTCCCTC AACGGGGTGT GGGGAGAACA 780 GTCATTTTTT TTTGAGAGAT CATTTAACATTTAATGAATA TTTAATTAGA AGATCTAAAT 840 GAACATTGGA AATTGTGTTC CTTTAATGGTCATCGGTTTA TGCCAGAGGT TAGAAGTTTC 900 TTTTTTGAAA AATTAGACCT TGGCGATGACCTTGAGCAGT AGGATATAAC CCCCACAAGC 960 TT 962 4118 base pairs nucleic acidsingle linear Genomic DNA 17 GGGCGAATTG GCGGCCGCGG GTTGCGGAGG GTGGGCCTGGGAGGGGTGGT GGCCATTTTT 60 TGTCTAACCC TAACTGAGAA GGGCGTAGGC GCCGTGCTTTTGCTCCCCGC GCGCTGTTTT 120 TCTCGCTGAC TTTCAGCGGG CGGAAAAGCC TCGGCCTGCCGCCTTCCACC GTTCATTCTA 180 GAGCAAACAA AAAATGTCAG CTGCTGGCCC GTTCGCCTCCCGGGGACCTG CGGCGGGTCG 240 CCTGCCCAGC CCCCGAACCC CGCCTGGAGC CGCGGTCGGCCCGGGGCTTC TCCGGAGGCA 300 CCCACTGCCA CCGCGAAGAG TTGGGCTCTG TCAGCCGCGGGTCTCTCGGG GGCGAGGGCG 360 AGGTTCACCG TTTCAGGCCG CAGGAAGAGG AACGGAGCGAGTCCCGCCGC GGCGCGATTC 420 CCTGAGCTGT GGGACGTGCA CCCAGGACTC GGCTCACACATGCAGTTCGC TTTCCTGTTG 480 GTGGGGGGAA CGCCGATCGT GCGCATCCGT CACCCCTCGCCGGCAGTGGG GGCTTGTGAA 540 CCCCCAAACC TGACTGACTG GGCCAGTGTG CTGCAAATTGGCAGGAGACG TGAAGGCACC 600 TCCAAAGTCG GCCAAAATGA ATGGGCAGTG AGCCGGGGTTGCCTGGAGCC GTTCCTGCGT 660 GGGTTCTCCC GTCTTCCGCT TTTTGTTGCC TTTTATGGTTGTATTACAAC TTAGTTCCTG 720 CTCTGCAGAT TTTGTTGAGG TTTTTGCTTC TCCCAAGGTAGATCTCGACC AGTCCCTCAA 780 CGGGGTGTGG GGAGAACAGT CATTTTTTTT TGAGAGATCATTTAACATTT AATGAATATT 840 TAATTAGAAG ATCTAAATGA ACATTGGAAA TTGTGTTCCTTTAATGGTCA TCGGTTTATG 900 CCAGAGGTTA GAAGTTTCTT TTTTGAAAAA TTAGACCTTGGCGATGACCT TGAGCAGTAG 960 GATATAACCC CCACAAGCTT GAGTATTCTA TAGTGTCACCTAAATAGCTT GGCGTAATCA 1020 TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCACAATTCCACA CAACATACGA 1080 GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAGTGAGCTAACT CACATTAATT 1140 GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGTCGTGCCAGCT GCATTAATGA 1200 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGCGCTCTTCCGC TTCCTCGCTC 1260 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGGTATCAGCTCA CTCAAAGGCG 1320 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAAAGAACATGTG AGCAAAAGGC 1380 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGGCGTTTTTCCA TAGGCTCCGC 1440 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGAGGTGGCGAAA CCCGACAGGA 1500 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCGTGCGCTCTCC TGTTCCGACC 1560 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGGGAAGCGTGGC GCTTTCTCAT 1620 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTCGCTCCAAGCT GGGCTGTGTG 1680 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCGGTAACTATCG TCTTGAGTCC 1740 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCACTGGTAACAG GATTAGCAGA 1800 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGTGGCCTAACTA CGGCTACACT 1860 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAGTTACCTTCGG AAAAAGAGTT 1920 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCGGTGGTTTTTT TGTTTGCAAG 1980 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATCCTTTGATCTT TTCTACGGGG 2040 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTTTGGTCATGAG ATTATCAAAA 2100 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTTTTAAATCAAT CTAAAGTATA 2160 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCAGTGAGGCACC TATCTCAGCG 2220 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCGTCGTGTAGAT AACTACGATA 2280 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATACCGCGAGACCC ACGCTCACCG 2340 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGGCCGAGCGCAG AAGTGGTCCT 2400 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCCGGGAAGCTAG AGTAAGTAGT 2460 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTACAGGCATCGT GGTGTCACGC 2520 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAACGATCAAGGCG AGTTACATGA 2580 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTCCTCCGATCGT TGTCAGAAGT 2640 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCACTGCATAATTC TCTTACTGTC 2700 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACTCAACCAAGTC ATTCTGAGAA 2760 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAATACGGGATAA TACCGCGCCA 2820 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTTCTTCGGGGCG AAAACTCTCA 2880 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCACTCGTGCACC CAACTGATCT 2940 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAAAAACAGGAAG GCAAAATGCC 3000 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATACTCATACTCTT CCTTTTTCAA 3060 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCGGATACATATT TGAATGTATT 3120 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCCGAAAAGTGCC ACCTGACGTC 3180 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATAGGCGTATCAC GAGGCCCTTT 3240 CGTCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGACACATGCAGCT CCCGGAGACG 3300 GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAGCCCGTCAGGG CGCGTCAGCG 3360 GGTGTTGGCG GGTGTCGGGG CTGGCTTAAC TATGCGGCATCAGAGCAGAT TGTACTGAGA 3420 GTGCACCATA TGCGGTGTGA AATACCGCAC AGATGCGTAAGGAGAAAATA CCGCATCAGG 3480 CGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTAAATATTTGTT AAATCAGCTC 3540 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTATAAATCAAAAG AATAGACCGA 3600 GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCACTATTAAAGA ACGTGGACTC 3660 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGCCCACTACGTG AACCATCACC 3720 CAAATCAAGT TTTTTGCGGT CGAGGTGCCG TAAAGCTCTAAATCGGAACC CTAAAGGGAG 3780 CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTGGCGAGAAAGG AAGGGAAGAA 3840 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCGGTCACGCTGC GCGTAACCAC 3900 CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCCATTCGCCATT CAGGCTGCGC 3960 AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTATTACGCCAGCT GGCGAAAGGG 4020 GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGTTTTCCCAGTC ACGACGTTGT 4080 AAAACGACGG CCAGTGAATT GTAATACGAC TCACTATA4118 4118 base pairs nucleic acid single linear Genomic DNA 18GGGCGAATTG GCGGCCGCGG GTTGCGGAGG GTGGGCCTGG GAGGGGTGGT GGCCATTTTT 60TGTCTAACCC TAACTGAGAA GGGCGTAGGC GCCGTGCTTT TGCTCCCCGC GCGCTGTTTT 120TCTCGCTGAC TTTCAGCGGG CGGAAAAGCC TCGGCCTGCC GCCTTCCACC GTTCATTCTA 180GAGCAAACAA AAAATGTCAG CTGCTGGCCC GTTCGCCTCC CGGGGACCTG CGGCGGGTCG 240CCTGCCCAGC CCCCGAACCC CGCCTGGAGC CGCGGTCGGC CCGGGGCTTC TCCGGAGGCA 300CCCACTGCCA CCGCGAAGAG TTGGGCTCTG TCAGCCGCGG GTCTCTCGGG GGCGAGGGCG 360AGGTTCACCG TTTCAGGCCG CAGGAAGAGG AACGGAGCGA GTCCCGCCGC GGCGCGATTC 420CCTGAGCTGT GGGACGTGCA CCCAGGACTC GGCTCACACA TGCAGTTCGC TTTCCTGTTG 480GTGGGGGGAA CGCCGATCGT GCGCATCCGT CACCCCTCGC CGGCAGTGGG GGCTTGTGAA 540CCCCCAAACC TGACTGACTG GGCCAGTGTG CTGCAAATTG GCAGATCGAT GACCTAAGTG 600GATCCGACTT GGTACCATGA ATGGGCAGTG AGCCGGGGTT GCCTGGAGCC GTTCCTGCGT 660GGGTTCTCCC GTCTTCCGCT TTTTGTTGCC TTTTATGGTT GTATTACAAC TTAGTTCCTG 720CTCTGCAGAT TTTGTTGAGG TTTTTGCTTC TCCCAAGGTA GATCTCGACC AGTCCCTCAA 780CGGGGTGTGG GGAGAACAGT CATTTTTTTT TGAGAGATCA TTTAACATTT AATGAATATT 840TAATTAGAAG ATCTAAATGA ACATTGGAAA TTGTGTTCCT TTAATGGTCA TCGGTTTATG 900CCAGAGGTTA GAAGTTTCTT TTTTGAAAAA TTAGACCTTG GCGATGACCT TGAGCAGTAG 960GATATAACCC CCACAAGCTT GAGTATTCTA TAGTGTCACC TAAATAGCTT GGCGTAATCA 1020TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA 1080GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT 1140GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA 1200ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC 1260ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 1320GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 1380CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 1440CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA 1500CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 1560CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT 1620AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 1680CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC 1740AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 1800GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 1860AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 1920GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 1980CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 2040TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 2100AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 2160TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG 2220ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 2280CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG 2340GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 2400GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 2460TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC 2520TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA 2580TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 2640AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC 2700ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 2760TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA 2820CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 2880AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT 2940TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 3000GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 3060TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT 3120TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC 3180TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT 3240CGTCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG 3300GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG 3360GGTGTTGGCG GGTGTCGGGG CTGGCTTAAC TATGCGGCAT CAGAGCAGAT TGTACTGAGA 3420GTGCACCATA TGCGGTGTGA AATACCGCAC AGATGCGTAA GGAGAAAATA CCGCATCAGG 3480CGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATATTTGTT AAATCAGCTC 3540ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 3600GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 3660CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC 3720CAAATCAAGT TTTTTGCGGT CGAGGTGCCG TAAAGCTCTA AATCGGAACC CTAAAGGGAG 3780CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA 3840AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC 3900CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCC ATTCGCCATT CAGGCTGCGC 3960AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT TACGCCAGCT GGCGAAAGGG 4020GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT 4080AAAACGACGG CCAGTGAATT GTAATACGAC TCACTATA 4118 975 base pairs nucleicacid single linear Genomic RNA 19 GGGCGAAUUG GCGGCCGCGG GUUGCGGAGGGUGGGCCUGG GAGGGGUGGU GGCCAUUUUU 60 UGUCUAACCC UAACUGAGAA GGGCGUAGGCGCCGUGCUUU UGCUCCCCGC GCGCUGUUUU 120 UCUCGCUGAC UUUCAGCGGG CGGAAAAGCCUCGGCCUGCC GCCUUCCACC GUUCAUUCUA 180 GAGCAAACAA AAAAUGUCAG CUGCUGGCCCGUUCGCCUCC CGGGGACCUG CGGCGGGUCG 240 CCUGCCCAGC CCCCGAACCC CGCCUGGAGCCGCGGUCGGC CCGGGGCUUC UCCGGAGGCA 300 CCCACUGCCA CCGCGAAGAG UUGGGCUCUGUCAGCCGCGG GUCUCUCGGG GGCGAGGGCG 360 AGGUUCACCG UUUCAGGCCG CAGGAAGAGGAACGGAGCGA GUCCCGCCGC GGCGCGAUUC 420 CCUGAGCUGU GGGACGUGCA CCCAGGACUCGGCUCACACA UGCAGUUCGC UUUCCUGUUG 480 GUGGGGGGAA CGCCGAUCGU GCGCAUCCGUCACCCCUCGC CGGCAGUGGG GGCUUGUGAA 540 CCCCCAAACC UGACUGACUG GGCCAGUGUGCUGCAAAUUG GCAGAUCGAU GACCUAAGUG 600 GAUCCGACUU GGUACCAUGA AUGGGCAGUGAGCCGGGGUU GCCUGGAGCC GUUCCUGCGU 660 GGGUUCUCCC GUCUUCCGCU UUUUGUUGCCUUUUAUGGUU GUAUUACAAC UUAGUUCCUG 720 CUCUGCAGAU UUUGUUGAGG UUUUUGCUUCUCCCAAGGUA GAUCUCGACC AGUCCCUCAA 780 CGGGGUGUGG GGAGAACAGU CAUUUUUUUUUGAGAGAUCA UUUAACAUUU AAUGAAUAUU 840 UAAUUAGAAG AUCUAAAUGA ACAUUGGAAAUUGUGUUCCU UUAAUGGUCA UCGGUUUAUG 900 CCAGAGGUUA GAAGUUUCUU UUUUGAAAAAUUAGACCUUG GCGAUGACCU UGAGCAGUAG 960 GAUAUAACCC CCACA 975 975 base pairsnucleic acid single linear Genomic RNA 20 GGGCGAAUUG GCGGCCGCGGGUUGCGGAGG GUGGGCCUGG GAGGGGUGGU GGCCAUUUUU 60 UGUCUAACCC UAACUGAGAAGGGCGUAGGC GCCGUGCUUU UGCUCCCCGC GCGCUGUUUU 120 UCUCGCUGAC UUUCAGCGGGCGGAAAAGCC UCGGCCUGCC GCCUUCCACC GUUCAUUCUA 180 GAGCAAACAA AAAAUGUCAGCUGCUGGCCC GUUCGCCUCC CGGGGACCUG CGGCGGGUCG 240 CCUGCCCAGC CCCCGAACCCCGCCUGGAGC CGCGGUCGGC CCGGGGCUUC UCCGGAGGCA 300 CCCACUGCCA CCGCGAAGAGUUGGGCUCUG UCAGCCGCGG GUCUCUCGGG GGCGAGGGCG 360 AGGUUCACCG UUUCAGGCCGCAGGAAGAGG AACGGAGCGA GUCCCGCCGC GGCGCGAUUC 420 CCUGAGCUGU GGGACGUGCACCCAGGACUC GGCUCACACA UGCAGUUCGC UUUCCUGUUG 480 GUGGGGGGAA CGCCGAUCGUGCGCAUCCGU CACCCCUCGC CGGCAGUGGG GGCUUGUGAA 540 CCCCCAAACC UGACUGACUGGGCCAGUGUG CUGCAAAUUG GCAGAUCGAU UCCCGUCGCC 600 AAAUCGAGCG GGUACCAUGAAUGGGCAGUG AGCCGGGGUU GCCUGGAGCC GUUCCUGCGU 660 GGGUUCUCCC GUCUUCCGCUUUUUGUUGCC UUUUAUGGUU GUAUUACAAC UUAGUUCCUG 720 CUCUGCAGAU UUUGUUGAGGUUUUUGCUUC UCCCAAGGUA GAUCUCGACC AGUCCCUCAA 780 CGGGGUGUGG GGAGAACAGUCAUUUUUUUU UGAGAGAUCA UUUAACAUUU AAUGAAUAUU 840 UAAUUAGAAG AUCUAAAUGAACAUUGGAAA UUGUGUUCCU UUAAUGGUCA UCGGUUUAUG 900 CCAGAGGUUA GAAGUUUCUUUUUUGAAAAA UUAGACCUUG GCGAUGACCU UGAGCAGUAG 960 GAUAUAACCC CCACA 975 975base pairs nucleic acid single linear Genomic RNA 21 GGGCGAAUUGGCGGCCGCGG GUUGCGGAGG GUGGGCCUGG GAGGGGUGGU GGCCAUUUUU 60 UGUCUAACCCUAACUGAGAA GGGCGUAGGC GCCGUGCUUU UGCUCCCCGC GCGCUGUUUU 120 UCUCGCUGACUUUCAGCGGG CGGAAAAGCC UCGGCCUGCC GCCUUCCACC GUUCAUUCUA 180 GAGCAAACAAAAAAUGUCAG CUGCUGGCCC GUUCGCCUCC CGGGGACCUG CGGCGGGUCG 240 CCUGCCCAGCCCCCGAACCC CGCCUGGAGC CGCGGUCGGC CCGGGGCUUC UCCGGAGGCA 300 CCCACUGCCACCGCGAAGAG UUGGGCUCUG UCAGCCGCGG GUCUCUCGGG GGCGAGGGCG 360 AGGUUCACCGUUUCAGGCCG CAGGAAGAGG AACGGAGCGA GUCCCGCCGC GGCGCGAUUC 420 CCUGAGCUGUGGGACGUGCA CCCAGGACUC GGCUCACACA UGCAGUUCGC UUUCCUGUUG 480 GUGGGGGGAACGCCGAUCGU GCGCAUCCGU CACCCCUCGC CGGCAGUGGG GGCUUGUGAA 540 CCCCCAAACCUGACUGACUG GGCCAGUGUG CUGCAAAUUG GCAGAUCGAU CGUCCAAUCG 600 CUAUACUCUCGGUACCAUGA AUGGGCAGUG AGCCGGGGUU GCCUGGAGCC GUUCCUGCGU 660 GGGUUCUCCCGUCUUCCGCU UUUUGUUGCC UUUUAUGGUU GUAUUACAAC UUAGUUCCUG 720 CUCUGCAGAUUUUGUUGAGG UUUUUGCUUC UCCCAAGGUA GAUCUCGACC AGUCCCUCAA 780 CGGGGUGUGGGGAGAACAGU CAUUUUUUUU UGAGAGAUCA UUUAACAUUU AAUGAAUAUU 840 UAAUUAGAAGAUCUAAAUGA ACAUUGGAAA UUGUGUUCCU UUAAUGGUCA UCGGUUUAUG 900 CCAGAGGUUAGAAGUUUCUU UUUUGAAAAA UUAGACCUUG GCGAUGACCU UGAGCAGUAG 960 GAUAUAACCCCCACA 975

What is claimed is:
 1. A method for the detection of metastatic tumorcells in a blood sample, comprising: (a) removing the non tumor cellsfrom the sample; (b) specifically amplifying the RNA component oftelomerase in the sample; and (c) determining quantitatively the amountof amplified nucleic acid to thereby detect the presence of tumor cellsin the blood sample, wherein the detection of tumor cells is indicativeof metastasis.
 2. The method of claim 1, wherein prior to theamplification of the RNA, the RNA contained in the sample is reversetranscribed into cDNA.
 3. The method of claim 2, wherein prior toreverse transcription of the RNA, the sample is treated with DNase. 4.The method of claim 1, wherein prior to step (b), the RNA contained inthe sample is purified.
 5. The method of claim 4, wherein purificationis effected by ion exchange chromatography.
 6. The method of claim 4,wherein purification is effected on silica gel.
 7. The method of claim1, wherein, for quantitative determination of the amplified nucleicacid, at least one, optionally three, standard nucleic acids arecoamplified and are added in different concentrations to the sample. 8.The method of claim 7, wherein the coamplifying standard nucleicacids(s) comprise one or more of the sequences of nucleotides set forthin any of FIGS. 7, 8 and
 9. 9. The method of claim 7, wherein,quantification is effected by comparing the amount of coamplifiednucleic acid or nucleic acids with the amount of the amplified nucleicacid.
 10. The method of claim 1, wherein the amplified nucleic acids arequantified either directly or via a label.
 11. The method of claim 10,wherein the label is selected from the group consisting of a radioactivelabel, a biotin label, a fluorescent label and anelectrochemoluminescent label.
 12. The method of claim 1, wherein theamplified nucleic acids are detected via a hybridization with a labeledoligonucleotide.
 13. The method of claim 12, wherein the label isselected from the group consisting of a radioactive label, a biotinlabel, a fluorescent label and an electrochemoluminescent label.
 14. Themethod of claim 1, wherein the sample is peripheral blood, and wherein anucleic acid, different from the amplified nucleic acid and known to bepresent in peripheral blood, is specifically coamplified with the RNAcomponent and detected as a positive control for the amplification ofthe RNA component.
 15. The method of claim 14, wherein the nucleic acidthat is amplified as a positive control is selected from mRNA coding forβ-globin and glyceraldehyde-phosphate dehydrogenase.
 16. The method ofclaim 1, wherein, as a negative control, no reverse transcriptionreaction is carried out before the amplification reaction with thesample and/or water is employed in place of the blood.
 17. The method ofclaim 1, wherein the following oligonucleotide primers are used for theamplification: 5′ GACTCGGCTC ACACATGCAG TTCGC 3′ (TM1) and/or (SEQ IDNO. 1) 5′ CTGGTCGAGA TCTACCTTGG GAGAAGC 3′ (TM2) (SEQ ID NO. 2)


18. The method of claim 17, wherein TM1 and/or TM2 comprises an RNApolymerase promoter.
 19. The method of claim 1, wherein a DNA polymeraseor an RNA polymerase is used for the amplification.
 20. The method ofclaim 19, wherein, amplification with DNA polymerase is effected by thepolymerase chain reaction (PCR) and, amplification with RNA polymeraseis effected by isothermal nucleic acid sequence-based amplification(NASBA).
 21. The method of claim 1, wherein any tumor cells in the bloodsample are concentrated.
 22. The method of claim 21, whereinconcentration is effected by immunoabsorption.
 23. The method of claim1, wherein the amount of amplified nucleic acid is determined in avenous blood sample and in an arterial blood sample, and the results arecompared with one another.
 24. The method of claim 1, wherein: theamount of amplified nucleic acid is determined in a blood sample from afinger pad, and in a venous or arterial blood sample, and the resultsare compared with one another.
 25. The method of claim 1, wherein thetumor cells are metastases of malignant tumors.
 26. The method of claim25, wherein the tumor cells are micrometastases.
 27. The method of claim1, wherein the tumor cells are from metastasizing tumors and/orneoplasms, from a T-cell lymphoblastoma, T-cell leukemia cells, chronicmyeloid leukemia cells, acute lymphatic leukemia cells, chroniclymphatic leukemia cells, tetratocarcinoma, melanoma, carcinoma of thelung, large intestine cancer, breast cancer, hepatocellular carcinoma,kidney tumor, adrenal tumor, prostate carcinoma, neuroblastoma, braintumor, rhabdomyosarcoma, leilomyasarcoma and lymphoma.
 28. The method ofclaim 1, wherein removal of non tumor cells is effected byimmunoabsorption.
 29. The method of claim 1, further comprisingquantification of any tumor cells in the sample.
 30. The method of claim1, wherein the detection of tumor cells is indicative of incipientmetastasis.
 31. The method of claim 1, wherein the detection of tumorcells is effected before organ metastases become manifest.
 32. Themethod of claim 1, wherein the detection of tumor cells is indicative ofthe efficacy of cancer therapy.
 33. The method of claim 1, wherein thenon tumor cells are stem cells and/or activated immune cells.